The objective of this proposal is to characterize the signaling roles of two candidate heparan sulfate proteoglycan (HSPG) modifying enzymes, quail glucosamine 6-O-sulfatase 1 (QSulf1) and QSulf2 in developing neural tube. Cell surface HSPGs play important roles in modulating the distribution and/or activity of signaling molecules such as Wnts, Shh, FGFs and BMPs. QSulf1, the first known extracellular matrix G6-sulfatase, is required for somite MyoD activation through its enzymatic activity to regulate Wnt pathway. QSulf2 is the second member of the family with unknown function. QSulf1 and QSulf2 have overlapping and discrete expression domains in embryonic neural tube. The proposal will test the hypothesis that QSulf1 and QSulf2 have unique functions in extracellular signaling pathways and the functional distinction is controlled by their divergent hydrophilic domains. First, we will characterize the expression pattern of QSulf1 and QSulf2 during embryogenesis by whole mount in situ hybridization combined with immunohistochemistry. Results from this experiment will serve as the initial steps for the functional analysis of QSulfs in developing neural tube. Second, to define QSulf function in the neural tube, we will manipulate the expression of each gene both by electroporation-mediated overexpression and antisense inhibition in chick embryos at stage 12. Molecular markers for neural tube patterning and neural crest cells will be characterized by immunohistochemistry. Results from these experiments will provide information in signaling roles of QSulfs in embryonic neural tube. Third, co-immunoprecipitation and yeast two-hybrid screen will be conducted to identify the binding proteins for the divergent hydrophilic domains of QSulfs that are hypothesized to control the localized functions of QSuIf1 or QSulf2. This study will provide insights on the mechanisms for localized QSulf function in the embryo.